Staphylococcus aureus and methicillin resistance detection directly from pediatric samples using PCR assays with differential cycle threshold values for corroboration of methicillin resistance

Abstract Staphylococcus aureus is a major human pathogen, causing a variety of nosocomial and community-acquired infections. While S. aureus usually grows well, there are situations where it cannot be isolated in culture, such as patients who have received prior antimicrobial therapy. There are commercially available tests for molecular identification of S. aureus and methicillin resistance; however, they often have limited utility due to restrictive specimen requirements, lack of data in pediatric populations and issues with specificity for methicillin resistance detections. Our objective was to evaluate the performance of laboratory-developed PCR assays that detect S. aureus and methicillin resistance directly from various specimen types. We developed two real-time PCR assays: 1) a singleplex assay targeting the nucA gene and 2) a multiplex PCR assay (mecA/SCC-orf PCRs) that detects the mecA gene and the conjunction region where SCC mec elements insert into the genome. A total of 538 pediatric specimens, including specimens from the lower respiratory tract ( n  = 149), abscess/wounds ( n  = 245), tissue and body fluids ( n  = 144), were tested and the results compared with culture and susceptibility testing. The nucA PCR is sensitive and specific for detection of S. aureus when compared with culture with an overall agreement of 93.1% and sensitivity and specificity of 93.5% and 93.0%, respectively. Among those culture-confirmed and nucA PCR positive specimens ( n  = 145), concordance between mecA/SCC-orf PCRs, using cycle threshold values for corroboration, and conventional methods was 98.6% and the sensitivity and specificity were 97.3% and 100%, respectively. The assays' performance suggests they are rapid, reliable tools to detect and differentiate between methicillin susceptible and methicillin resistant S. aureus in our pediatric patient population providing diagnostic impact when used in conjunction with culture.